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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

An OECD 421 for read-across substance, 4 -methyl-4 phenylpentan-2 -ol, is available. On the basis of the available study data, the No Observed Adverse Effect Level (NOAEL) of the read-across substance, 4-methyl-4 phenylpentan-2-ol was considered to be 400 mg/kg/day. Taking into account the molecular weight correction between source and target substance (178.27 vs. 220.31), the NOAEL for 1,3-Dimethyl-3-phenylbutyl acetate is 494.32 mg/kg.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 October - 26 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Department of Safety Assessment Advinus Therapeutics Limited Bangalore 560 058, India
- Age at study initiation: (P) 11 wks;
- Weight at study initiation: (P) Males:G1 : 268.95 ± 13.19 G2 : 274.08 ± 16.14 G3 : 273.73 ± 14.48 G4 : 270.52 ± 17.02 ;
Females: G1 : 200.24 ± 14.71 G2 : 198.28 ± 10.05 G3 :96.55 ± 15.00 G4 :196.41 ± 11.29
- Fasting period before study:
- Housing:Rats were housed under standard laboratory conditions, air conditioned with adequate fresh air supply (12-15 air changes/hour).
The maximum and minimum temperature in the experimental room was recorded once daily.
The relative humidity in the experimental room was calculated daily from dry and wet bulb temperature recordings.
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum):Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet - Pellet (Certified) manufactured by Harlan Laboratories B.V. Maasheseweg 87c PO Box 553, 5800, AN Venray, The Netherlands, was provided ad libitum
- Water (e.g. ad libitum): purified deep bore well water ad libitum
- Acclimation period:After successful clinical examination for good health, the rats were acclimatized for five days before start of the treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):20-24 °C,
- Humidity (%):relative humidity 59 – 66%, .
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 hours light and dark cycle

IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dose formulation were prepared one day prior to commencement of treatment,the same was used on day 1 of treatment and thereafter dose formulations were prepared daily or a day in advance, extemporaneously and used within the prescribed stability (8 days) period.

VEHICLE
- Justification for use and choice of vehicle (if other than water): suspension in corn oil
- Concentration in vehicle: 0, 8 , 24 or 80 mg/mL
- Amount of vehicle (if gavage): 5mL/kg
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: not stated
- Proof of pregnancy: sperm in vaginal smear referred to as gestation day 0
- After successful mating each pregnant female was caged singly.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Males: The dose formulations were administered daily, starting 2 weeks prior to mating and continued during the mating period and until approximately 80% of the females had delivered.
Females: The dose formulations were administered daily, starting 2 weeks prior to the mating period, during the mating and pregnancy period until lactation day 3.
Frequency of treatment:
Daily
Details on study schedule:
Females were placed with males from the same group in a 1:1 ratio.
Cohabitation was continued until evidence of sperm in the vaginal smear and /or vaginal plug. Subsequently, mated females were housed individually until lactation day 4. The day of confirmed mating was designated as Gestation Day 0 (GD0). The pre-coital time was calculated for each mated female confirmed through vaginal smear.
Remarks:
Doses / Concentrations:
0, 40, 120 or 400 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Positive control:
No
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily - twice daily during treatment period for morbidity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily - Physical or gross behavioural abnormalities observed in dams and offspring recorded

BODY WEIGHT: Yes
- Time schedule for examinations: day 1 of treatment and at necropsy


OTHER:
Food consumption of pregnant dams was recorded on GD 0-7, 7-14 , 14-20 and 0-4 of lactation
Duration of gestation measured

Sperm parameters (parental animals):
Parameters examined in P male parental generations:

testis weight, epididymis weight - these tissues were also preserved for microscopic examination. Prostate also preserved for microscopic examination.

Histological examination of testes included a qualitative assessement of stages of sprematogenesis.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:

Mean litter size per group
Live birth index
Day 4 survival index
Malformations
Gross pathological lesions


GROSS EXAMINATION OF DEAD PUPS:
yes, for possible defects and possible cause of death.
Postmortem examinations (parental animals):
SACRIFICE
-All male rats were sacrificed after approximately 80% of females have delivered.
-Pregnant females sacrificed on day 4 of lactation
-Live pups were sacrificed on Lactation Day 4 along with respective dams.
-Females which have not delivered on day 25 post-coitum were sacrificed on day 25 post-coitum


GROSS NECROPSY
-Gross necropsy consisted of examinations by veterinary pathologist. Females - numbers of implantation sites and corpora lutea
-Dead and necropsied pups wre examined for malformations and or cause of death


HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared for microscopic examination respectively.
Tissues were preserved in 10% neutral buffered formalin for histopathological examination.
Epididymides (also weighed)
Ovaries
Oviduct
Prostate
Seminal vesicles and coagulating glands
Testes (preserved in Davidsons fluid) (also weighed)
plus any tissue with gross lesions
Postmortem examinations (offspring):
GROSS NECROPSY
-Dead and necropsied pups wre examined for malformations and or cause of death

Statistics:
The statistical analysis of the experimental data was carried out using the validated package in Excel and also using licensed copies of SYSTAT
Statistical package ver.12.0. All quantitative variables like body weight, food intake, organ weights and organ weight ratios were tested for normality
(Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modeling by treatment
groups. Non-optimal (non-normal or heteroscedastic) data were transformed, before using ANOVA. Means between treatment and control groups
were compared using Dunnett’s test if the overall ‘F’ test found to be significant.
Pre-implantation loss (%), post implantation loss (%), no. of corpora lutea,implantations, pre-coital interval and gestation length (days) were analysed
after suitable transformation (√ x + ½) of the data. One-way analysis of variance (ANOVA) was carried out for the transformed data. Dunnett’s
pairwise comparison of the treated means with the control mean was done for the significant group differences. Z test was performed for testing the differences in proportions for mating and fertility indices.
All analyses and comparisons were evaluated at the 5% (P≤0.05) level.
Statistically significant differences (P≤0.05) were identified
Reproductive indices:
Female fertility index (%)
Mean number of corpora lutea (CL)/group
Mean number of implantations/group
Implantation index
Percentage of pre-implantation loss per group
Post implantation loss (%)
Gestation index
Offspring viability indices:
Mean litter size per group
Live birth index (%)
Day 4 survival index (%)
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
No treatment related mortality or clinical signs were observed at any of the doses tested. A single incidence of local alopecia in the back region noticed in female animal No. Ro2121 at 0 mg/kg/day was considered as incidental.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
Males: No significant changes in body weight were observed when compared with control means.
Females: No significant changes in body weight were observed prior to the mating period when compared with control means.

In males, no significant change in food intake was observed when compared with control means.
In females, no significant change in food intake was observed when compared with control means.

No significant changes in maternal body weights were observed when compared with control means. A significant decrease in body weight change observed during gestation interval day 14-20 at 120 mg/kg/day was not associated with any change in the corresponding maternal food intake and hence, considered toxicologically insignificant.

No treatment-related change in maternal food intake was observed at all the doses tested, when compared with control means.

No significant changes in maternal body weight and food consumption were observed during different intervals of the lactation period when compared with control means.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
No treatment-related effects were observed with respect to mean pre-coital time, gestation length and fertility indices when compared to control. The
statistically significant decrease in male and female fertility and female fecundity indices observed at 40 mg/kg/day can be ascribed to pregnancy
failure in single female (Ro2149) and, hence, was considered toxicologically not relevant.

No treatment-related effects were observed with respect to mean number of implantations and percentage of post implantation losses when compared to control means. However, a significant decrease in the percent implantation index observed at 120 mg/kg/day was contributed by the significant increase in the percentage of pre-implantation loss. The percentage pre-implantation loss was higher than the historical controls and was not increased at 400 mg/kg/day compared with the control group. Hence, was considered incidental due to lack of dose-response relationship.

ORGAN WEIGHTS (PARENTAL ANIMALS):
There were no treatment-related findings.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Gross examination of the adult rats, did not reveal toxicologically relevant lesions in any dose group.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Microscopic examination revealed no toxicologically relevant test item-related changes in the reproductive organs of males and females.

OTHER FINDINGS (PARENTAL ANIMALS)
Key result
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: The dose level indicated is the highest dose level used in the study based on previous dose range finding assessment
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
VIABILITY (OFFSPRING)
No significant changes in mean total number of pups and mean body weight of total pups per litter were observed on Lactation Days 0 and 4 when compared with control. However, a significant decrease in the mean total number of pups per litter observed at 120 mg/kg/day on Lactation Day 4 was presumed to be contributed by pup mortality/cannibalism during day 0 through day 4. This effect was within the historical controls (Annexure 8) and, hence, not considered to be toxicologically relevant.

No treatment-related effects on the mean litter size and mean viable litter size were observed when compared to control means. No external abnormalities
were detected in live or dead pups in any of the groups. The Day 4 survival index was significantly lower at 120 mg/kg/day, which was considered
toxicologically insignificant due to lack of dose-response relationship.


BODY WEIGHT (OFFSPRING)
No significant changes in mean number and mean body weight of male or female pups per litter were observed on Lactation Days 0 and 4 when
compared with control.




Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
viability
Remarks on result:
other:
Remarks:
This was the highest parental dose used in the study determined from previous dose range finding studies
Reproductive effects observed:
not specified
Conclusions:
On the basis of the available study data, the No Observed Adverse Effect Level (NOAEL) of the read-across substance 4-methyl-4-phenylpentan-2-ol, was considered to be 400 mg/kg/day after daily oral (gavage) administration to male and female Wistar rats for a period of 6 weeks in males and about 7 weeks in females.
Executive summary:

Daily oral (gavage) administration of the read-across substance, 4-methyl-4 phenylpentan-2-ol at doses of 40, 120 and 400 mg/kg/day to male and female Wistar rats for specified duration was well tolerated. No test item-related mortality or clinical signs occurred. Body weight, food consumption, pre-coital time, gestation length, mating and fertility parameters, number of implantations, percentage of pre- and post implantation losses, mean litter size and mean viable litter size as well as testes and epididymides weights were not affected by the treatment. External evaluation of pups as well as gross and microscopic examination of the reproductive organs of the parent animals revealed no adverse test item-related changes. However, a significant decrease in the mean total number of pups per litter observed at 120 mg/kg/day on Lactation Day 4 was presumed to be contributed by pup mortality/cannibalism during day 0 through day 4. This effect was within the historical controls and, hence, not considered to be toxicologically relevant. A significant decrease in the percent implantation index observed at 120 mg/kg/day was contributed by the significant increase in the percentage of pre-implantation loss. The percentage pre-implantation loss was higher than the historical controls and was not increased at 400 mg/kg/day compared with the control group. Hence, was considered incidental due to lack of dose-response relationship. On the basis of the available study data, the No Observed Adverse Effect Level (NOAEL) of the read-across substance, 4-methyl-4 phenylpentan-2-ol

was considered to be 400 mg/kg/day after daily oral (gavage) administration to male and female Wistar rats for a period covering pre-mating, mating and post-mating, i.e. approximately about 6 weeks in males and about 7 weeks in females.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
494.32 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Short description of key information:
Daily oral (gavage) administration of the read-across substance, 4-methyl-4 phenylpentan-2-ol, at doses of 40, 120 and 400 mg/kg/day to male and female Wistar rats for specified duration was well tolerated. No test item-related mortality or clinical signs occurred. Body weight, food consumption, pre-coital time, gestation length, mating and fertility parameters, number of implantations, percentage of pre- and post implantation losses, as well as testes and epididymides weights were not affected by the treatment. Gross and microscopic examination of the reproductive organs of the parent animals revealed no adverse test item-related changes. However, the significant decrease in the mean total number of pups per litter on Lactation Day 4 and a significant decrease in the percent implantation index observed at 120 mg/kg/day were not increased at 400 mg/kg/day compared with the control group. Hence, was considered incidental due to lack of dose-response relationship. On the basis of the available study data, the No Observed Adverse Effect Level (NOAEL) on fertility for the read-across substance, 4-methyl-4 phenylpentan-2-ol, was considered to be 400 mg/kg/day after daily oral (gavage) administration to male and female Wistar rats for a period covering pre-mating, mating and post-mating, i.e. approximately about 6 weeks in males and about 7 weeks in females.

Effects on developmental toxicity

Description of key information
Daily oral (gavage) administration of the read-across substance, 4-methyl-4 phenylpentan-2-ol, at doses of 40, 120 and 400 mg/kg/day to male and female Wistar rats for specified duration was well tolerated. Following mating,gestation and parturition, the mean litter size and mean viable litter size were not affected by the treatment. External evaluation of pups revealed no adverse test item-related changes. 
On the basis of the available study data, the No Observed Adverse Effect Level (NOAEL) on rat pup development for the read-across substance, 4-methyl-4 phenylpentan-2-ol, was considered to be 400 mg/kg/day after daily oral (gavage) administration to male and female Wistar rats for a period covering pre-mating, mating and post-mating, i.e. approximately about 6 weeks in males and about 7 weeks in females.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
494.32 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Justification for selection of Effect on developmental toxicity: via oral route:
Limited developmental parameters were assessed in the key reproduction study

Justification for classification or non-classification

The absence of adverse effects in this study with the read-across substance, 4-methyl-4 phenylpentan-2-ol, indicates that the 1,3 -dimethyl-3 -phenylbutyl acetate shall not be classified as reproductive toxicant according to Regulation (EC) No 1272/2008 (2017).